The economic success of a commercial dairy operation is greatly influenced by the reproductive performance of the herd. For maximum profit and production, a cow should be pregnant by 90 days after calving, resulting in an average herd calving interval of 12.5 months. Less than optimum performance may be attributed to cows not cycling regularly, cows cycling but exhibiting poor signs of estrus and the stockman's failure to efficiently detect the cows in estrus. In some herds, 10 to 30% of cows in estrus escape detection and 10 to 25% of cows presented for insemination are not in estrus. Approximately 7% of pregnant cows may exhibit some signs of estrus. Insemination of such animals may cause termination of the existing pregnancy and wastage of the semen. Thus, accurate diagnosis of pregnancy, estrus and other related conditions is a valuable tool in animal management.
Progesterone is a steroidal hormone produced by the corpus luteum during the estrous cycle and pregnancy. Plasma progesterone levels accurately reflect different stages of the estrous cycle as well as the pregnancy status. Since progesterone levels in milk correlate well with those of plasma, milk progesterone tests have been extensively used to determine pregnancy, confirm estrus, diagnose reproductive disorders and monitor the effectiveness of infertility treatments in cows. Quantification of milk progesterone levels has been performed by radio immunoassay (RIA), a technique which requires expensive equipment and handling of hazardous radioactive materials. Another technique is the enzyme immunossay (EIA) wherein an enzyme bound to progesterone competes with the progesterone contained in the sample to be analyzed, the results being determined by using the enzyme to effect a color change proportional to the enzyme amount. Descriptions of such assays may be found in the following articles: W. F. Cleere et al. in the Irish Veterinary Journal, Vol. 39, pages 6-14 (1985); M. J. Sauer et at. in the British Vet. Journal, Vol. 138, pages 522-532 (1982); and C. F. Chang et al. in Steroids, Vol. 41, No. 2, pages 173-195 (1983).
However, even the most recent assay procedures described in the literature do not satisfy essential criteria to allow use of the test by technicians, usually dairymen, at the location of the cow, i.e., "cow-site", with test results available in a short period, e.g. less than 15 to 20 minutes. Of course any test which could be operated at cow-site, as opposed to a laboratory procedure, should also be accurate and indicative of a wide range of conditions in the cow, i.e., should convey as much information as possible and as accurately as possible. Another requirement of a test which would enable it to be a cow-site test is that the measurements which must be taken should not be so fine as to involve careful and meticulous physical operations. Thus, a practical cow-site test for progesterone would not involve manipulation of minute quantities, e.g. 10 to 50 microliters, of sample or reagent as is necessitated in prior assay procedures. It is thus an object of the present invention to provide an assay procedure for progesterone in a mammalian body fluid such as milk, serum or plasma which is accurate and indicative of a wide range of conditions, yet simple, fast and tolerant of differential amounts of reagents and samples used therein. That is, one or several microliters too much or too little reagent and/or sample should preferably have no discernible effect on the assay results.